Diverse cellular phenotypes and functions in metazoans are specified by differential expression of genes. Regulation of quantitatively precise expression of genes in the right cell type and at the fight time is mediated by the combinatorial and synergistic (or antagonistic) action of a limited number of transcription factors. Nrl, first identified by the PI, is a key basic motif-leucine zipper (bZIP) transcription factor, which is now established as a prime mediator of gene regulation in both developing and mature rod photoreceptors. Nrl interacts with Crx and other transcription regulatory proteins to synergistically (or antagonistically) modulate the expression of rhodopsin and many rod-specific genes. Mutations in the human NRL gene are associated with retinal degenerative diseases. We have shown that the deletion of Nrl by gene targeting in mice (Nrl -/-) results in complete lack of rod function and rod-specific gene expression; instead, there is enhanced S-cone function indicating a phenotypic transformation. Using the Nrl-promoter to drive enhanced green fluorescent protein (EGFP) in transgenic mice, we have demonstrated that Nrl expression during development corresponds to the genesis of rods. Continued expression of Nrl in mature rods suggests that it also plays a major role in maintaining appropriate expression of genes required for rod function. Our studies have identified Nr2e3 (photoreceptor specific nuclear receptor, PNR) as a direct transcriptional target of Nrl and revealed that Nr2e3 acts synergistically with Nrl and Crx in regulating rod phototransduction genes. In this renewal application, we propose to decipher the Nrl-mediated transcriptional regulatory network(s) in developing and mature rod photoreceptors. The specific aims of the project are as follows: Specific Aim 1: We will identify the genes that are directly regulated by Nrl ("direct targets") using a comprehensive strategy, involving microarray analysis, chromatin immunoprecipitation (CHIP) and promoter activity assays. Specific Aim 2: We will identify transcriptional regulatory proteins that interact with Nrl during early and late stages of rod development and validate the physiological relevance of selected interactions. Specific Aim 3: We will determine whether Nrl is sufficient to induce rod-specific gene expression and generate functional rods, by expressing Nrl in the Nrl -/- retina at temporally distinct stages. Specific Aim 4: We will delineate the function of Nr2e3, a direct target of Nrl, in developing and mature rod photoreceptors. Specific Aim 5: We will define the molecular mechanism(s) that underlie the pathogenesis of retinopathies, caused by mutations in the human NRL and NR2E3 genes. Since mutations in Nrl, its interacting proteins, and their target genes result in retinopathies, it is expected that our studies will reveal significant new insights into retinal diseases. A better understanding of transcriptional regulatory pathways may allow us to experimentally manipulate the expression of specific target gene(s) to correct a disease phenotype. [unreadable] [unreadable] [unreadable]